Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.